Phospholipase Cγ2 (PLCγ2) is a protein expressed predominantly in hematopoietic cells and has a great importance in immunoreceptor signaling. Our group previously found that PLCγ2 deficient mice were protected from autoantibody-mediated skin blistering model induced by the systemic injection of antibodies against type VII collagen (C7), which is located in the basement membrane. The inflammation mediated mainly by neutrophils was completely abrogated in PLCγ2 deficient mice, but the mechanism is presently unknown. We aimed to examine the expression of PLCγ2 in the main circulating leukocyte populations and investigate the in vitro activation of PLCγ2 deficient neutrophils by C7-anti-C7 immune complexes. Expression of PLCγ2 in circulating leukocyte populations was measured by flow cytometry after intracellular labelling. We used bone marrow derived neutrophils to examine in vitro cell activation plated onto immobilized C7-anti-C7 immune complex surfaces. Type VII collagen or anti-C7 served as control surfaces. Cell spreading was evaluated using phase-contrast microscopy. Production of reactive oxygen species (ROS) was measured in a cytochrome-C based reduction assay. PLCγ2 was present in neutrophils, eosinophils, monocytes and B cells but not in T cells of the wild type mice. While examining the effector functions of neutrophils activated by C7-anti-C7 immune complexes, we found that nearly a third of the cells of the wild type mice spread and a great amount of ROS was produced. On the contrary, both neutrophil spreading and ROS production were completely absent in immune complex-treated PLCγ2 deficient cells. Our results show that the PLCγ2 is expressed in the major myeloid cell populations-expression of the circulating immune cells and plays a crucial role in the spreading and ROS-production of neutrophils activated by C7-anti-C7 immune complexes in vitro, which can explain why PLCγ2 deficient mice are protected in autoantibody mediated skin blistering.

ESDR225 - The role of PCγ2 in the in vitro activation of neutrophils by type VII collagen immune complexes

50th Annual Meeting of the European Society for Dermatological Research. VIRTUAL MEETING